Dissection of physiological, transcriptional, and metabolic traits in two tall fescue genotypes with contrasting drought tolerance

Abstract Tall fescue (Festuca arundinacea) is an important cool‐season perennial forage grass that forms mutualistic symbioses with fungal endophytes. Physiological, biochemical and transcriptional comparisons were made between two tall fescue genotypes with contrasting drought tolerance (tolerant, T400, and sensitive, S279), either with or without endophyte (Epichloë coenophiala). Drought stress was applied by withholding watering until plants reached mild, moderate and severe stresses. Physiological characterization showed that T400 had narrower, thicker leaves, and lower leaf conductance under well‐watered conditions, compared to S279. After severe drought and recovery, endophytic T400 had greater shoot and root biomass than other plant types. Under drought, leaf osmotic pressure increased much more in T400 than S279, consistent with accumulation of metabolites/osmolytes, especially proline. Gene Ontology enrichment analysis indicated that T400 had more active organic acid metabolism than S279 under drought, and implicated the role of endophyte in stimulating protein metabolism in both genotypes. Overall T400 and S279 responded to endophyte differently in aspects of physiology, gene transcription and metabolites, indicating plant genotype‐specific reactions to endophyte infection.

Drought is the most important environmental factor limiting agriculture (Farooq et al., 2012). Plant responses to drought stress are complex and vary over space and time. Lack of water is perceived, in part, by membrane sensors in the root, which trigger systemic signaling pathways that affect gene expression throughout the plant. Plants have evolved diverse strategies to survive periods of drought, including developmental escape and avoidance, and biochemical tolerance (Fang & Xiong, 2015;Hirayama & Shinozaki, 2010;Meena & Kaur, 2019).
Drought resilience involves multiple traits, each typically controlled by multiple genes, which presents a major challenge for researchers and plant breeders interested in the underlying mechanisms and harnessing them to increase crop drought tolerance.
Previous studies have identified common transcriptional responses to drought in various species, including induction of genes involved in transcriptional regulation, photosynthesis, hormone especially abscisic acid (ABA) metabolism, antioxidant biosynthesis, and metabolism of carbohydrate, amino acids, and fatty acids (Benny et al., 2019;Egea et al., 2018;Wang et al., 2017). Changes in both primary and secondary metabolites are associated with drought responses.
Previous studies also point to important roles of osmolytes (e.g., trehalose, fructan and proline) as osmoprotectants under drought stress, among which, the importance of proline has been confirmed by various genetic studies (reviewed in Meena & Kaur, 2019).
Despite the importance of tall fescue as a primary forage species and of drought as a key limitation on forage production, the molecular and genetic mechanisms of drought tolerance in tall fescue remain largely unknown. Previous studies have explored physiological, biochemical and root developmental aspects of drought responses in tall fescue (Chen et al., 2018;Pirnajmedin et al., 2015;Saha et al., 2015;Sarmast et al., 2015;Sun et al., 2013) and the influence of endophytes on stress tolerance (Nagabhyru, Dinkins, Wood, et al., 2013). Only a few studies identified over/under-expressed transcripts responsive to drought stress (Dinkins et al., 2019;Talukder et al., 2015). A systems study on the drought tolerance mechanism and the role of endophyte in tall fescue drought responses is still lacking.
Here, we investigated the physiological, biochemical, and transcriptional responses to drought stress of two tall fescue genotypes contrasting in drought tolerance, which were selected based on field and preliminary greenhouse experiments. We also determined the impact of symbiosis with the endophyte, E. coenophiala on drought responses in the two plant genotypes. We aimed to understand the systems mechanisms underlying the variance in tall fescue drought tolerance and the role of endophyte in this process.

| Plant material and growth conditions
The tall fescue genotypes T400 and S279 were selected from the tall fescue cultivar Texoma MaxQ II. Texoma MaxQ II is a tall fescue cultivar developed by the Samuel Roberts Noble Foundation in cooperation with AgResearch Ltd for its improved persistence and yield, and superior adaptation to the south-central USA (Hopkins et al., 2011). In an earlier study exploring the genetic diversity related to drought tolerance of this cultivar, 1000 genotypes (plants) of Texoma MaxQ II were screened for drought tolerance in a greenhouse experiment using relative water content (RWC) and leaf osmotic potential (OP) as selection criteria. After initial screening, 25 selected most drought tolerant and susceptible genotypes were further evaluated in Ardmore, Oklahoma field for 2 years. These genotypes were also screened with PEG8000 in a greenhouse. Based on RWC and OP data obtained across all experiments, T400 and S279 were selected for its tolerance "T" and susceptibility "S" to drought stress, respectively, and used in the current study.
For T400 and S279, as well as all plants of Texoma MaxQ II, an asymptomatic novel fungal endophyte (E. coenophiala), AR584, lives in the intercellular space within the pseudostems (leaf sheath whorls). The endophyte makes a stable symbiosis with the plant and transmitted through seed. To generate endophyte-free plants, the inherent AR584 endophyte in those genotypes was removed using a hydroponic system as described in Nagabhyru, Dinkins, Wood, et al. (2013). Both the developed clonal pairs were confirmed for endophyte status using immunoblot  and polymerase chain reaction assays (Takach et al., 2012).
Tall fescue plants were propagated by tillers and planted in tall plastic cones (35 × 7 cm, D60L, Stuewe and Sons., Inc.; https://www. stuewe.com). The soil was a mixture of metromix 360 and common sand (v/v = 2/1). At planting, two tillers were planted in each pot. The soil water content was monitored with EC-5 soil sensors (https://www.meter group.com/). For uniformity, the top edge of the soil sensor was 20 cm to the soil upper surface in each pot.

| Drought treatment and plant sampling
Three weeks after being planted into the soil, ¾ of the plants were subjected to water withholding (drought-stressed) and ¼ plants remained well-watered (control). Drought-stressed plants were harvested when the soil volumetric water content (VWC) reached 10% (mild-stressed DrtA), 5% (moderately-stressed, DrtB), and 1% (severely-stressed, DrtC), respectively. The VWC of well-watered plants (Ctl) were maintained at ~30%. To minimize variance, all samples were harvested between 1 pm and 2 pm each day. At harvest, the shoots and roots were collected separately and then frozen in liquid nitrogen immediately. The tissues were stored at −80°C until being ground in liquid nitrogen for RNA purification (RNAseq) and metabolite analysis gas chromatography-mass spectrometry (GC-MS). For tissue collection to be used in quantification of the leaf OP, shoot/root dry weight and other physiological parameters, a separate drought experiment was performed.

| Drought and re-watering experiment
In a separate experiment from above, two tillers of each of the four plant types were planted in a three-gallon plastic pot, two centimeters away from the edges avoiding the center of the pot, in random orders. A total of four pots and eight tillers of each plant type were used. When the soil VWC decreased to less than 1% and all the leaves lost chlorophyll and dried out, each pot was re-watered and the plants were allowed to re-grow for 20 days. At the end of re-growth, shoots and roots were harvested separately and dried completely in a 55°C oven for dry weight quantification.

| Leaf size and specific leaf weight
The size of the youngest fully-expanded leaf of a tall fescue plant was measured with a Li-3000A portable area meter (Li-Cor; https:// www.licor.com/). After area measurement, the leaf was completely dried in a 55°C oven and then the dry weight was quantified using a lab balance. The leaf specific weight was calculated by dividing the leaf area by the dry weight.

| Guard-cell density
The youngest fully-expanded leaf of a tall fescue plant at harvest was collected and nail polish imprints were made of the middle session of the leaf, avoiding the edges. The imprints were subsequently observed and photographed under a microscope (Nikon TE300) at 100×. Stomata density was counted from photos.

| Leaf conductance
Leaf conductance was measured with the SC-1 Leaf Porometer (METER Group, Inc.; https://www.meter group.com/) on the youngest fully-expanded leaf. Each leaf was measured twice at the middle session and the average value was used.

In-vivo leaf chlorophyll content was measured with a Chlorophyll
Meter SPAD-502plus (Spectrum Technologies; http://www.specm eters.com/) on the youngest fully-expanded leaf. Each leaflet was measured two times at the middle session and the average reading was used. The leaf edges were avoided at all measurements.

| Leaf OP
The middle section (1 cm) of the youngest fully-expanded leaf was sampled and then fully hydrated in sterile and de-ionized water in a 2 ml Eppendorf tube for 48 h at 4°C. Next, the fully-hydrated leaves were tap-dried on a filter paper to remove surface water, and then stored at −80°C for over 24 h in a 0.65 ml Eppendorf tube. At the end of storage, a hole was punched at the bottom of the 0.65 ml Eppendorf tube and then it was placed inside a 1.5 ml Eppendorf tube, being centrifuged at 16,000 g for 10 min at 4°C to collect the leaf sap. The molal concentration of the leaf sap was measured at room temperature with a Wescor EliTechGroup Vapro 5600 Vapor Pressure Osmometer. Osmotic potential was calculated using the formula "OP = iCRT", where i = ionization constant, C = Molal concentration (mole/kg), R = pressure constant (0.0831 liter bar/mole °K), T = temperature °K (273+°C).

| Transcriptome analysis with RNAseq
Total RNA was isolated with the Spectrum™ Plant Total RNA Kit All sequences were first quality trimmed using a custom Perl script which removed low quality bases (quality score < 30) from the ends of reads until two consecutive high-quality bases were found.
Reads less than 30 bp long after trimming were discarded, along with their mate pair. Each sample was then de novo assembled with Trinity version 2.2.0 (https://github.com/trini tyrna seq/trini tyrnaseq).
These independent assemblies were then merged by selecting sample CTL400P-SH1 as a starting set, aligning the next (in alphabetical order) assembly with it using BLASTN, and adding the transcripts for which no homologs were found to the starting set. This process was repeated for every other sample, adding the unique transcripts from every other assembly to the starting set. Each sample's reads were

| Metabolite analysis with GC-MS
Metabolite analysis of polar and non-polar metabolites were conducted following the procedure in Kang et al. (2011). Data analysis was performed using software MS-DIAL (http://prime.psc.riken.jp/ Metab olomi cs_Softw are/MS-DIAL/).

| Proline biochemical assay
Proline content was analyzed with a biochemical assay following Bates et al (Bates et al., 1973) and Hamid et al (Hamid et al., 2003).
Proline concentration was determined using a standard curve generated using L-proline.

| Statistical analysis
For phenotypic and leaf osmotic pressure data, significant analysis was performed in R with package "agricolae". Two-way ANOVA (aov) was performed first and then Duncan's New Multiple Range Test was conducted for p value calculations. For GC-MS and RNAseq data, significant analysis was performed by calculating p values with student's t test (two tails assuming equal variance) in excel. False discovery rate (FDR) adjusted p values (p adj ) were calculated in R using function "fdr".

| Bioinformatic analysis
For having the best annotations, all bioinformatic analyses were performed using the closest Arabidopsis thaliana orthologs of correspond-

| Physiological characterization of drought adaptation traits
Before performing drought stress experiments, we compared the shoot, root, and leaf phenotypes of the drought-tolerant tall fescue genotype, T400, and the drought-sensitive genotype, S279 under well-watered conditions, with (E+) or without endophyte (E−). Shoot  Figure S1). On the other hand, no significant difference was observed between T400E+ and T400E− in either shoot or root biomass (Figure 1a,b).
Leaf size and thickness of well-watered plants were then compared. T400 had relatively long, narrow, and thick leaves, whereas S279 leaves were shorter, wider, and thinner (Figure 1c-g). The area of each leaf was similar among all plant-endophyte combinations ( Figure 1c). The difference between E+ and E− was not significant.
Stomatal density on the abaxial side of leaves was similar among different plant types ( Figure S2), while leaf conductance was significantly higher in S279 compared to T400 (Figure 1h). Leaf rolling was first evident at soil VWC of 5% (moderate stress, DrtB) and reached an extreme at 1% soil VWC (severe stress, DrtC; Figure 2). Under well-watered conditions, T400E+ had the highest leaf chlorophyll content with 48.5 SPAD units, which was 18.2% higher than that of S279E+ with the lowest chlorophyll content. In addition, endophyte infection significantly reduced leaf chlorophyll content by 12% in S279, but did not cause significant changes in T400 ( Figure 3a). Under severe drought stress, leaf chlorophyll content significantly decreased in S279E− but not in other plant types ( Figure 3a). T400 and S279 had similar leaf OP at well-watered conditions ( Figure 3b). Under severe drought stress, the leaf OP of T400 was 19% (E+) to 24% (E−) higher than that of well-watered controls, whereas no significant difference was observed in S279E+/− between drought and well-watered conditions ( Figure 3b). After severe drought stress, all plant types had similar shoot biomass, while S279E-had smaller root biomass than S279E+, which was similar to T400E+/− ( Figure S3).
In a separate experiment, the four tall fescue plant types were planted together in three-gallon pots and the shoot/root biomass was measured after severe drought stress (<1% soil VWC) and recovery ( Figure 4). After 20 days of recovery and re-growth, T400E+ had much larger shoot and root biomass compared to other plant types, especially root biomass, which was nearly twice that of other plant types. No significant difference was observed between S279E+ and S279E−, either in shoot or root (Figure 4b,c).   Table S1).

| Transcriptomic and metabolomic analyses of well-watered and drought-stressed tall fescue plants
RNA-seq analysis was carried out for plants with or without endophyte exposed to different levels of drought stress, to identify genes and associated biological processes affected by drought. Under drought stress, there were generally more downregulated than up-regulated genes, and more differentially expressed genes (DEGs) in the roots than shoots (Figure 6a,d,e). The difference in transcript regulation between T400 and S279 was minimal under moderate stress (DrtB; Figure 6b). Numbers of DEGs between E+ and E− plants were much smaller compared to that between T400 and S279, showing generally higher transcript levels in E+ than E− plants, especially in the shoot (Figure 6c). The endophyte effect on root gene expression was very small in both T400 and S279 ( Figure 6c).

Gene Ontology (GO) enrichment analysis was performed on
drought-regulated genes, which revealed that the following processes were induced under drought stress in all plant types: response to abiotic stresses (temperature, heat, high light, desiccation, salinity, cold, oxidative) and catabolism of organic acids, amino acids, cofactors, porphyrin-containing compounds, and tetrapyrrole/chlorophyll. In contrast, genes associated with photosynthesis, biotic stress response (chitin), growth (response to nitrogen), receptor signaling pathways, phosphorylation and phosphate metabolism were substantially repressed under drought conditions (Table S2).
Next, GO enrichment analyses were performed on genes that were differentially expressed in T400 and S279, and between E+ and E−. When looking at the GO enrichment of genes that were differentially expressed in T400E− and S279E−, which presumably reflect intrinsic genetic differences between T400 and S279, five categories of genes were found to be enriched in the shoot, but none in the root (Table S3). Enriched genes involved in (programmed) cell death were generally more highly expressed in T400 shoots than in S279, under both well-watered and stressed conditions. On the other hand, enriched genes related to response to chitin and nitrogen compound typically had lower expression levels in T400 shoots than in S279 under well-watered conditions (  (Table S3).
When combining the genotype and endophyte effects and comparing between T400E+ and S279E+, we found that genes involved in degradation of organic acids and amino acids were enriched among the genes that had higher expression levels in drought-stressed T400E+ than S279E+ in the shoot, but no significant category enrichment was identified in up-regulated genes (T400E+/S279E+) in roots (Table S3). In contrast, strong enrichment was observed in the shoot down-regulated genes (T400E+/S279E+), many in categories related to photosynthesis activity that responded to drought stress, i.e. light reaction, porphyrin-containing compound biosynthesis/ metabolism, tetrapyrrole biosynthesis/metabolism, photosynthetic electron transport, plastid organization, and chlorophyll biosynthesis (Table S3).

| D ISCUSS I ON
Plant drought adaptation and resistance include three main strategies: drought escape, drought avoidance, and drought tolerance (Aslam et al., 2015;Levitt, 2015). Drought escape refers to plants that alter their life cycle by either entering dormancy or flowering early when faced with drought stress (Kramer, 1980). Drought avoidance is related to a plant's ability to maintain high water potential under water limitation, mostly by reducing leaf transpiration and/ or enhanced root growth (Levitt, 2015). In contrast, plant droughttolerance is primarily related to maintaining water uptake by accumulating osmolites under drought stress (Levitt, 2015). Earlier studies indicate that tall fescue uses all three strategies to survive drought stress. It is well known that Mediterranean tall fescue can enter summer dormancy in dry and hot environments, which is a typical mechanism of drought escape (Volaire & Norton, 2006). Under drought, tall fescue plants tend to develop deeper roots and larger root systems, an important mechanism for drought survival that was shown repeatedly to be associated with drought tolerance among different varieties (Carrow, 1996;Huang & Fry, 1998;Pirnajmedin et al., 2015). Past studies also showed that drought tolerant tall fescue cultivars contain higher protein and soluble carbohydrate content, and lower H 2 O 2 content than sensitive ones (Rohollahi et al., 2018). For osmotic adjustment, multiple studies reported sharp increase of proline in tall fescue leaves under drought stress Pirnajmedin et al., 2017;Rohollahi et al., 2018;Sarmast et al., 2015). The role of other osmolytes such as sugar alcohols were much less studied (Bacon, 1993).
In the current study, we compared drought responses of two contrasting tall fescue genotypes and found that the drought tolerant genotype, T400, showed morphological and physiological characteristics related to drought avoidance and are typical for plants that are adapted to dry environments, e.g. small, narrow, but thick leaves, and relatively lower leaf conductance compared to the sensitive genotype, S279 (Figure1). This phenomenon has been reported broadly in grasses and other plant species, and these plants are generally called "water savers" (Kang et al., 2011;Maricle et al., 2007;Polania et al., 2016). Although leaf traits of E+ and E− plants were similar in both T400 and S279, endophyte symbiosis affected plant biomass differently in T400 and S279. Under both well-watered ( Figure 1) and drought ( Figure S3) conditions, S279E+ plants had significantly higher root biomass but similar shoot biomass compared to S279E−. In T400, endophyte infection did not promote root growth significantly under either conditions (Figure 1; Figure S3). However, F I G U R E 4 Tall fescue plants (a), shoot (b) and root (c) dry weight after severe drought stress (soil VWC < 1%) and recovering. Different letters indicate significant difference at p < .05 (Duncan's test), n = 5, error bars are standard errors. Image of plants in a pot is shown in (a). VWC, volumetric water content [Colour figure can be viewed at wileyonlinelibrary. com]

(a) (b) (c)
TA B L E 1 Major polar metabolite accumulation in severely drought-stressed (DrtC) compared to well-watered plants (Ctl). Log2 fold changes (FC) of metabolites are shown, with up-and down-regulated metabolites colored in red and blue, respectively. FCs less than 1.5 (−0.85 < log 2 ratio < 0.85) are not shown. All FCs having p values less than .1 but equal to or larger than .05 are in bold only and FCs with p < .05 are in bold and underlined, n = 3 Group Metabolite ID  S279E+ S279E-T400E+ T400E-S279E+ S279E-T400E+ T400E In earlier studies, endophyte symbiosis has been shown to promote plant growth and improve drought resistance (Feng et al., 2006;Khan et al., 2014). In tall fescue, endophyte presence was reported to increase shoot biomass, tiller numbers, and survival under field drought stress, while the benefit was not noticeable during wet years (West et al., 1993). In another study using three tall fescue genotypes and multiple endophyte species, significant genotype × endophyte interactions (p < .001) were observed for tiller density and shoot dry weight per area, indicating the promoting effect of endophyte on plant growth is association-specific (Elbersen & West, 1996). Similar tall fescue cultivar × endophyte interaction was found in a separate study with elite cultivars infected with elite endophytes performing the best, and endophyte was more important in conferring resistance than difference between cultivars (Hume & Sewell, 2014). Therefore, the interaction between specific tall fescue and endophyte genotype appears to be important for the outcome. Here, we demonstrate that T400 and S279 responded to the same endophyte infection differently at the levels of phenology, physiology, molecular and biochemistry, and endophyte infection is crucial in enabling drought tolerance in T400, as discussed further below.
At molecular level, GO enrichment analysis revealed that genes related to photosynthesis were expressed at lower levels in T400E+ than in S279E+ (Table S3) Figure S1).
Under both well-watered and drought stressed conditions, T400E+ was much more active in protein biosynthesis and metabolism than T400E− (Table S3). Together, these observations may explain why T400E+ had the largest root and shoot biomass after severe drought stress and recovery (Figure 4). Our study confirms that the presence of endophyte has a positive effect on root growth and drought stress tolerance, as reported earlier in tall fescue (Arachevaleta et al., 1989;Bacon, 1993;West et al., 1993). In addition, T400 and S279 responded to endophyte differently in multiple levels (Figures 1, 3, and 4; Table 1), presumably due to plant genotype-specific reactions to endophyte infection as reported earlier in tall fescue (Elbersen & West, 1996;Hume & Sewell, 2014).
As mentioned above, we observed a significant difference between plant genotypes in leaf osmotic pressure changes during drought, with T400 having a much larger leaf osmotic pressure increase under drought stress compared to S279 (Figure 3b). Higher leaf osmotic pressure indicates stronger osmotic adjustment and more osmolite accumulation, which is crucial for surviving drought stress and has been reported in tall fescue (West et al., 1990). Compared  with drought-adaptive phenotypic changes, e.g. smaller and thinker leaves, and lower stomatal density, osmotic adjustment is inducible and temporary. Therefore, it generally has less negative effect on growth and is more cost-effective to plants (Johnson & Asay, 1993;McCree, 1986). Metabolite profiling confirmed greater accumulation of specific metabolites under severe drought stress in T400 than in S279 shoots (Table 1), especially organic acids. Consistent with this, GO enrichment analysis revealed genes involved in amino acid and organic acid catabolism amongst those with higher expression levels in T400 than S279 under drought stress (Table S3).
Among all metabolites detected, proline accumulated much more in T400 than in S279 under severe drought stress, in both roots and shoots, and both E+ and E− (Tables 1 and 2). In T400E+, proline content increased from 0.16 to 8.16 mg/g DW in the shoot, equivalent to a change in OP of 69.5 mmol/kg, explaining much of the leaf osmotic pressure increase under drought stress (Figure 3b).
Transcript levels of one of the two proline biosynthetic enzymes, P5CS, mirrored those of proline content, consistent with P5CS being a rate-limiting enzyme in proline biosynthesis (Delauney & Verma, 1993). Early studies demonstrated that over-expression of P5CS in multiple plant species promotes proline biosynthesis and improves drought tolerance (Amini et al., 2015;Kavi Kishor et al., 1995;Vendruscolo et al., 2007;Yamchi et al., 2007). Similar association between proline accumulation, P5CS induction, and genotype drought sensitivity was reported in rice (Choudhary et al., 2005), Brassica juncea (Phutela et al., 2000), and wheat (Maghsoudi et al., 2018). However, proline accumulation was found not to be associated with genotype drought tolerance in Arabidopsis (Marín-de la Rosa et al., 2019), alfalfa (Kang et al., 2011), and Tibetan hulless barley (Deng et al., 2013). Increased proline content does not necessarily associate with improved drought tolerance either (Pospisilova et al., 2011). Therefore, while proline is undoubtedly an important drought osmolite in plants, it may not be a universal marker for plant drought tolerance. In tall fescue, we observed contrasting patterns F I G U R E 6 Numbers of differentially expressed genes (DEGs; FC > 2, p adj < .05) that were regulated by drought stress (a), between T400 and S279 (b), and between E+ and E− (c). Severe drought stress (DrtC) regulated genes in shoots (d) and roots (e) are illustrated by a web-based illustration tool Divenn (https://divenn.tch.harva rd.edu/; Sun et al., 2019). Red denotes up-regulated genes; blue denotes downregulated genes, and yellow denotes up-or down-regulated genes [Colour figure can be viewed at wileyonlinelibrary.com] of proline accumulation associated with drought tolerance in the two genotypes, with more proline accumulated in the tolerant geno- type. An earlier study in tall fescue obtained similar results with tolerant cultivar 'Van Gogh' accumulating 32% more leaf proline than the sensitive cultivar 'AST7002' under drought (Man et al., 2011).
In the future, it would be interesting to expand this study to more genotypes and test the potential role of proline as a biochemical signature in screening for drought tolerance in tall fescue.

| SUMMARY
In summary, gradual soil drought stress was applied to two tall fescue genotypes (T400 and S279) with contrasting drought tolerance, either with or without endophyte symbiosis. Physiological and biochemical analysis indicate that T400 (tolerant genotype) utilizes both drought escape and drought tolerance strategies to confer greater drought tolerance than S279 (sensitive genotype), for example, thicker and narrower leaves, lower transpiration, and more osmoticum especially proline accumulation under drought stress.
Metabolite analysis with GC-MS identified common and unique metabolites altered by drought stress in T400 and S279, with or without endophyte symbiosis. GO enrichment analysis of transcriptome changes revealed that the drought tolerant genotype, T400, repressed more genes related to photosynthesis and induced more genes related to organic acid and amino acid metabolism than the sensitive genotype. GO enrichment analysis also highlighted the role of endophyte in stimulating protein biosynthesis and metabolism in both genotypes.

ACK N OWLED G EM ENTS
The authors would like to thank Konstantin Chekhovskiy, Jennifer

Black, Melissa McMahon, Alicia Maple, Behnam Khatabi, Yuhong
Tang, and Guifen Li for assistance with data collection and technical support, and thank Raul Huertas for critical suggestions on the data analysis. This work was supported by Noble Research Institute, LLC.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available